J. Zikán

Interactions of pig Fab γ fragments with protein a fromStaphylococcus aureus

Ninety % of pig serum IgG was bound to protein A-Sepharose. Both individual fractions of the IgG, separated on the basis of their electric charge, were adsorbed on protein A-Sepharose to a similar extent. However, these fractions differed in their elution profile from the protein A-Sepharose when a gradient of increasing molarity of MgCl2 was used. Relative amounts of fractions eluted in higher concentrations of MgCl2 were augmented with the increasing amount of IgG bound to SpA-Sepharose. Not only high proportions of Fc fragments, but also nearly half of Fab fragments reacted with protein A. This latter interaction, confirmed also by affinity electrophoresis, did not have the character of a specific reaction of antibody with antigen.

Interactions of pig Fab mu and Fab alpha fragments with protein A from Staphylococcus aureus.

Forty % of serum IgM and 47 % of dimeric colostral IgA were bound to protein A-Sepharose. Fab μ and Fabα fragments showed a reactivity similar to that of the whole immunoglobulins. Complete elutions of IgM and IgA from the protein A-Sepharose were obtained in lower concentrations of MgCl2 than the complete elution of IgG. However, IgM and IgA were completely eluted at higher concentrations of MgCl2 in the presence of IgG than in its absence.

Two structurally different types of rabbit light chains.

Disulphide bonds of rabbit γ-G-globulin and the antibody of the γ-G-globulin type against the 2,4-dinitrophenyl group were split both by the oxidative sulphitolysis at pH 8.6 and by the reduction with 2-mercaptoethanol followed by carboxymethylation. The fractionation was carried out in 0.05 m formic acid containing 6m urea, in 1m propionic acid or in 6m guanidine hydrochloride. Both heavy (H) and light) (L) chains are released from the I+J fraction preceding on an elution diagram H chains when rechromatographed in a stronger desaggregation medium. A small amount of the L chains is also released on rechromatography of the H chains (isolated from 1m propionic acid) in 6m guanidine hydrochloride. The separation of the degraded γ-G-globulin in 0.05m formic acid containing 6m urea or in 6m guanidine hydrochloride showed a separation of the L chains to two fractions differing by electrophoretic properties, peptide maps and N-terminal amino acids. However, these chains exhibit a similar molecular weight, immunoelectrophoretic behaviour and similar properties on reactivation of the antibody H chain.

Cross-reactivity of human and a putative pig IgD. Pig IgD-like molecules as serum and lymphocyte components.

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, the ratio of radioactivity of their heavy and light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.

Influence of proteolysis inhibitors and immunoadsorption technique on the composition of rabbit antigen-specific receptor preparations. Presence of Fc receptors

Addition of proteolysis inhibitors during the isolation procedure of rabbit arsanylated bovine IgG specific receptors decreased significantly the amount of low-molar-mass proteins in all receptor preparations.Rabbit ARS-BGG-specific receptor preparations isolated by immunoadsorption technique contain molecules which do not react with antigen and antibodies against immunoglobulins and have identical molar mass andchymotryptic peptide composition as those of isolated Fc receptors. It is suggested that during isolation of antigen-specific receptors from the surface of lymphoid cells, Fc receptors react with complexes composed of antigen and Ig+ receptors on the surface of immunoadsorbent and are isolated together with antigen-specific receptors.

The appearance of an IgD-like molecule on pig lymphocytes during ontogeny.

An IgD-hke molecule was found on the surface of pig fetal lymphocytes using guinea pig anti human IgD antisera and indirect immunofluorescence. The binding of an tiIgD antisera could be specifically inhibited with human IgD Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen) There is evidence for their endogenous origin. The number of lymphocytes bearing IgD like molecules rapidly increased during ontogeny.

Low molecular rabbit antibodies against 2,4 dinitrophynyl group

Rabbit antibody against 2,4 dinitrophenyl group with molecular weight 75,000 was isolated. Its binding activity was demonstrated polarographically and by passive haemagglutination. The antibody consisted of subunits which were connected only with non-covalent bonds. It is not clear so far if the antibody was produced by enzymatic splitting of antibodies of the IgG type or was synthetized in the form which is found in the serum.

Role of saccharides in immunoglobulin--Fc receptor interactions.

The rosettes formed by mouse peritoneal macropahges or DCH-5 cells and TNP-erythrocytes coated with anti-TNP antibodies of different isotypes were inhibited to various extent by monosaccharides. The most effective inhibitors were N-acetylglucosamine, glucosamine, mannose and N-acetylneuraminic acid in 1–5 mmol/L concentrations. Even more efficient were glycopeptides isolated from IgG molecules. The Fc receptors (FcRs) released from DCH-5 cells during cultivation and gradually separated by affinity chromatography on immobilized IgG reacted with aggregated IgG and inhibited the rosette formation. The FcRs eluted by monosaccharides influenced mainly the number of rosettes mediated by IgA and IgE while those eluted with a glycine-HCl buffer inhibited preferentially IgG rosettes. As shown by SDS-PAGE the heterogeneity of the fraction eluted with a mixture of monosaccharides revealed one main component with an effective molar mass of 50 kg/mol. The glycine-HCl eluate contained two major components of 55 and 38 kg/mol. The IgG-Sepharose 4B bound all the fractions but only the binding of the 50 kg/mol molecule could be inhibited by monosaccharides.

Heterogencity of human polyclonal IgE reacting with staphylococcal protein A

A small part of polyclonal IgE (6 %) was bound to protein A-Sepharose from the serum of M.P., containing a high concentration of IgE. No monoclonal IgE isolated from the serum of V.L. was bound to this sorbent. This binding of polyclonal IgE appears to be heterogeneous since a multiphasic pattern was observed with discontinuous pH gradient elution from protein A-Sepharose. Also, like IgE from the whole serum, monomeric IgE isolated from the serum of M.P. on Sepharose 6B showed this binding heterogeneity. It is suggested that IgE molecules with different affinities for protein A could belong to different isotypic or allotypic variants.

Changes in the level and character of colostral immunoglobulins during the lactation period in sows.

A profound decrease in the concentration of colostral proteins (among immunoglobulins primarily IgG) during the first three days of lactation is accompanied by changes in the molecular heterogeneity of IgA. In the course of lactation, the amount of 7S IgA increases in relation to 10S IgA. The total amount of IgA, after the initial decrease during the first three days, maintains a gradual increase. In addition, a transient increase of natural haemagglutinating antibodies toEscherichia coli (serotype O55) was found in fractions corresponding to IgA and IgG during lactation.